The goal of the proposed work is to develop heterobivalent agents for targeted delivery of imaging and therapy to metastatic melanoma. These agents contain two ligands that direct the construct to crosslink two different cell surface receptors, resulting in dramatic increases in binding selectivity. This has advantages over agents directed against single receptors in that they do not rely on overexpression of a single cell surface protein. The proposed constructs also have advantages over other multifunctional agents in that they are produced via convergent synthesis and are designed to be relatively small with favorable ADME characteristics. Work during the first period of support has (1) identified and validated a receptor combination that can be used to target a subset of metastatic melanomas, and (2) demonstrated a proof-of-principle that synthetic heterobivalent agents can crosslink heterologous receptors. These constructs bound with up to 50-fold higher affinity compared to corresponding monovalent interactions. Current research will combine these two advances to develop heterobivalent agents against an identified target receptor pair. To achieve this goal, Aim 1 will use a previously developed G-protein coupled receptor (GPCR) system to derive analytical solutions that predict behavior of multivalent ligands as imaging agents. Aim 2 will develop bivalent agents that target a receptor combination identified in the first period of support: N-formyl peptide receptor like, type 2 (FPRL2) and the type 1 melanocortin receptor (MC1R). In this aim, optimum linker and ligand chemistries will be determined with high-throughput binding assays. Bivalent ligands will be labeled with fluorophores for testing by in-cyto and in-vivo imaging. Those with optimum characteristics will be labeled with DOTA. DOTA derivatives will be used to chelate Eu for ex vivo fluorescence and 111In for in vivo SPECT assessment of pharmacokinetics and biodistribution. Aim 3 will continue the validation effort for 21 additional receptors that were identified as targets during the first period of support. These will be validated primarily though immunohistochemistry of tissue microarrays containing multiple melanoma grades as well as multiple normal tissues. It is expected that this work will result in the validation of additional receptor combinations that will target a majority of metastatic melanomas. At the end of this next period of support, we will have (1) developed more precise analytical methods to predict the behavior of multivalent ligands as imaging agents; (2) developed targeting ligands that will be useful against 40-50% of metastatic melanomas and (3) identified additional 3- and 4-receptor target combinations for the remaining molecular phenotypes of this disease.The goal of the proposed work is to develop platform targeting agents for delivery of imaging and therapy to metastatic melanoma. These agents are heterobivalent in that they link together two ligands that are directed against two different cell surface receptors. Such agents have advantages over agents directed against single epitopes by not relying on a single overexpressed cell surface protein. They also have advantages over other multifunctional agents in that they are produced via convergent synthesis and hence are relatively small with acceptable ADME characteristics.